Although many glutaraldehyde/surfactant solutions have been evaluated for their sporicidal or bactericidal activity, very few such solutions have been thoroughly investigated from the mycobactericidal activity view point. It is well-known that there is no direct relationship between spores and mycobacteria resistances to chemical disinfectants. For example, while under certain conditions phenols and isopropylalcohol can be extremely potent against M. tuberculosis, the same chemicals have always displayed very poor sporicidal activity.
It is also well-known that cells of M. tuberculosis are among the most resistant vegetative microorganisms, and after bacterial endospores, these cells constitute the most severe challenge to chemical germicides. However, until recently, the efficacy of glutaraldehyde solutions to quickly destroy M. tuberculosis on animate or inanimate surfaces has been questioned by several authors. Rubbo et al (J. Appl. Bact., 30:78-87, 1967) were the first to challenge the tuberculocidal efficacy of 2% alkaline glutaraldehyde solutions sold under the CIDEX trade name (Surgikos, a Johnson and Johnson company). A 1971 study by T. Bergan and A. Lysad relates to the antitubercular action of several types of disinfectants using a method adapted from the Kelsey-Sykes test for disinfectants. It was concluded that the 2% glutaraldehyde solution was not adequately effective, growth occurring after the second incremental addition of bacteria. In November 1976, Boucher et al filed a report at the Environmental Protection Agency on a comparative study of the tuberculocidal efficacy of acid and alkaline glutaraldehyde (2%) compositions. These conclusions, based on hundreds of Association of Official Analytical Chemists (AOAC) tests using more than 4000 test tubes conducted at the Ontario Research Foundation, -found- that 2% alkaline glutaraldehyde did not kill M. tuberculosis var bovis (BCG) in 10 or even 20 minutes at 20.degree. C. Although tuberculocidal times seemed to be shorter (15 to 20 min) with a potentiated acid glutaraldehyde at pH 3.5, results were not statistically significant. It was noted that the use of different neutralizers (sodium bisulfite or horse serum) provided different results. Following the identical method, slight variations of temperature between 20.degree. C. and 25.degree. C. also gave wide variations in tuberculocidal activity.
In November 1976, FM Collins and V. Montalbine reported (Journ Clin Microb, p. 408-412, Nov. 1976) that 2% alkaline glutaraldehyde solution inactivated 10.sup.5 viable M. tuberculosis H37Rv cells present on the surface of porcelain penicylinders within 3 min. at 18.degree. C. A potentiated acid glutaraldehyde needed five minutes to be tuberculocidal at the same temperature. This method was different from the AOAC procedure (a pass/fail test) and the results were so optimistic that they were skeptically received.
During this period, numerous discrepancies resulting from use of the AOAC method (or procedures derived from it) attracted the attention of many scientists interested in a more accurate methodology. In October 1984, J M Ascenzi, T M Wendt, and J W McDowell in a paper entitled "Important Information Concerning the Reuse of Glutaraldehyde-Based Disinfectants and their Tuberculocidal Activity", first evaluated seven known glutaraldehyde sterilizing compositions with a new quantitative technique said to be very accurate and reproducible. They varied the contact times (1, 2, 5, 10, and 20 minutes) at a standard temperature of 20.degree. C. and none of the disinfectants which were tested showed complete kill of the test populations of mycobacteria in ten or twenty minutes. Table I shows the specific data pertaining to this study. This was extremely important since all of these commercial solutions had been previously approved for 10 and 20 minutes tuberculocidal efficacy with the AOAC method. The Environmental Protection Agency (EPA), having regulation authority over label claims of sterilants and disinfectants, convened a panel of experts in September 1985 to evaluate this new quantitative procedure and to compare it with the old AOAC method. Notwithstanding, the EPA decided that all Registrants/Applicants of all antimicrobial pesticides with existing tuberculocidal claims for fresh or reused solutions would have to retest their products with one of the three following options:
(a) The new quantitative method, (b) the old AOAC method but with substantial modification of the exposure time and temperature, (c) the standard AOAC method using 20.degree. C. and 10 minutes exposure time in a laboratory other than the one which developed the original data.
The tuberculocidal data call-in-notice was issued on Jun. 13, 1986 and the results were released on Feb. 17, 1989. Only 43 products of a total of 144 satisfied the data requirements of the call-in. Among the glutaraldehyde-based products only 60% satisfied the call-in requirements. Only four companies tested their products with the new and more accurate quantitative method. Vast discrepancies exist in comparing the quantitative method with the old AOAC approach. For instance, at 20.degree. C., a standard alkaline solution (CIDEX 2%) needed 70 min. to kill M. tuberculosis, the CIDEX formula needed 2 hours and the CIDEX Machine 4 hours. Previously, these solutions were claiming a 10 minute kill time at 20.degree. C. with the AOAC method (J M Ascenzi et al, Surgikos, Res. Div., Oct. 1984). The influence of temperature is appreciated by the data showing 2% alkaline solution (CIDEX) kills M. tuberculosis in 70 min. at 20.degree. C., in 30 min. at 25.degree. C., and in only 10 min. at 30.degree. C. U.S. Pat. No. 3,917,850 shows that a mixture of glutaraldehyde and phenol (or phenate salt) in the presence of anionic compounds could display some mycobactericidal synergism.
The new quantitative procedure of J M Ascenzi, T M Wendt, and J W McDowell appears in the Environmental Protection Agency PR Notice 80-1 of May 28, 1986, Section 2, "Tuberculocidal Efficacy Testing". This utilizes a kill curve concept in which a suspension of approximately 10.sup.6 colony forming units (CFU) of M. bovis BCG is added to each ml of disinfectant in tubes held at the appropriate temperature.
Aldehydes have a strong odor and their vapors can be very irritating to mucous membranes. To solve this problem researchers have tried to combine aldehydes with various glycols (U.S. Pat. No. 3,886,269). Through hydrogen bonding, glycols and aldehydes form physical complexes (i.e., larger molecules) which exhibit a lower vapor pressure and less eye and skin irritation. This method was first suggested by Trujillo and Lindell in a paper entitled, "New Formaldehyde Based Disinfectants" (J. Appl. Microb, 26(1):106-110, July 1973). The same year, Harriet Field of the Queen Mary Veteran's Hospital in Montreal, Canada, reported the elimination of noxious glutaraldehyde vapors using propylene glycol and glycerol. The direct complexing of a glutaraldehyde solution with triethyleneglycol was first reported by Boucher in the summer of 1975. On Feb. 15, 1977, the first odorless commercial glutaraldehyde/triethylene glycol composition was approved by the USDA under the trade name AGROCIDE 2. A concentrate of this formula was later registered by the EPA (Feb. 2, 1979) under the Registration No. 15136-5. Between 1976 and 1977, H. D. Muller of the University of Georgia College of Agriculture released several reports describing the successful replacement of formaldehyde by the Boucher glutaraldehyde/triethylene glycol solutions for poultry hatcheries applications (Evaluation of AGROCIDE in a commercial broiler hatchery, Field Trial II, Oct. 20, 1976 by Harry D. Muller, Ext. Poultry Sci, University of Georgia, Athens, Ga.). The use of these triethylene glycol complexes in hospitals was later mentioned by Boucher in November, 1978 (Respiratory Care 23(11):1063-1072). The glutaraldehyde/triethylene glycol solutions of Boucher and Muller were potentiated with TERGITOL 15-S-12 a non-ionic surfactant. The original formula registered by the USDA in early 1977 has been marketed in this country since 1975 under the trade name AGROCIDE, MC25, WAVICIDE-06, and 05. In all these formulations the amount of deodorizing triethylene glycol (TEG) was six times higher than the concentration of dialdehyde. In other words, a 0.5% glutaraldehyde formula contained 3% TEG while a 0.25% contained 1.5% TEG.